The plant secondary metabolism represents an extraordinary array of various chemical compounds with potent biological activities. The properties of many plant metabolites (e.g. biocidal activity) are dictated by the sessile lifestyle and e.g. adaptive evolution of abiotic and biotic defense mechanisms. Although complex and diversified, most plant secondary metabolites are formed by a few biosynthetic pathways. For instance, flavonoids and anthocyanidins are mainly produced by modification of final products of the shikimate pathway (i.e. phenylalanine or tyrosine). The aromatic amino acids are modified by consequential activity of several enzymes such as phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase (4CL), and type III polyketide synthase to yield a core chemical structure of naringenin chalcone that can be further augmented to the respective phenylpropanoid-products (Figure 1). The focal enzymatic reaction of this biosynthesis is the ATP-dependent activation of trans-4-coumaric acid with coenzyme A (CoA) catalyzed by a 4CL enzyme (E.C. 6.2.1.12). The transformation of carboxylic acids into reactive CoA thioesters resembles the fatty acid metabolism that also relies on CoA as a molecular carrier. Mechanistically, the enzymatic reaction proceeds through formation of a 4-coumaroyl-adenylate intermediate with simultaneous release of pyrophosphate by hydrolysis of adenosine triphosphate (ATP), followed by the subsequent transfer of 4-coumaroyl moiety to the sulfhydryl group of CoA. Our project stems around discovery of novel coding sequences for 4CL ligases in noncanonical plants. For this purpose, an acyl-activating enzyme consensus motif is used as an identifier for the 4CL gene family. The putative coding sequences were extracted from genomic and transcriptomic information and produced as recombinant proteins in Escherichia coli. Comparing to other plants that tend to contain multiple 4CL isoenzymes, we aim to functionally characterize novel coding sequences that might encode for 4CL enzymes and therefore to complement the existing acid-CoA enzyme family.

In vitro characterization of novel plant carboxylic acid-coenzyme A ligases

ACS Spring 2022

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